Influenza virus is an RNA virus belonging to Orthomyxoviridae, and known to be divided into the type A, type B and type C.
Influenza virus has an envelope having the lipid bilayer membrane structure. The inner layer of the envelope is mainly composed of matrix protein, and RNP, which is a complex between RNA and protein. On the outer layer, influenza NA protein (neuraminidase) and influenza HA protein (hemagglutinin) (hereinafter referred to as “NA protein” and “HA protein”, respectively), which are the so called surface proteins, exist as projections.
Viruses belonging to the same type are further classified into plural subtypes and strains based on the antigenicities of HA protein and NA protein, respectively. As influenza vaccines, those wherein an adjuvant, antiseptic and/or the like were added to a stock solution containing NA protein and HA protein are commonly used.
Influenza virus to be used for influenza vaccines is cultured mainly in embryonated chicken eggs. Further, in recent years, methods for culturing influenza virus by animal cell culture systems have been established.
Since, in general, influenza virus obtained by these methods does not exist solely but exists together with cultured cells, impure proteins and the like, the influenza virus needs to be separated from culture liquid or the like. Separation and purification of influenza virus are carried out by ultracentrifugation, ultrafiltration, density gradient centrifugation and/or the like.
However, even in the influenza virus separated and purified by these methods, impure proteins derived from the host are often observed. In cases where an influenza vaccine containing such impure proteins is inoculated, side effects such as anaphylactic shock or Guillain-Barre syndrome may occur, so that further purification is required.
Known examples of the method for producing an influenza antigen include a method wherein a gene for producing a protein antigen contained in an influenza virus is incorporated into an animal cell or insect cell.
As an example of the method for purifying influenza virus, a method wherein influenza virus or an influenza virus antigen is purified using hydroxyapatite is already known (Patent Document 1). The method according to Patent Document 1 comprises a step of adsorption of virus or a virus antigen to hydroxyapatite and a step of elution by an eluent. However, since various strains of influenza virus exist and the sequences of HA protein and NA protein vary among the strains, the elution position may vary among the influenza virus strains under the constant elution conditions, leading to different purities among the strains. Therefore, it is necessary to strictly control the conditions of elution of the adsorbed protein for each influenza virus strain, which is very complicated in view of production.
In Patent Document 2, a method for purifying a recombinant HA protein using a carrier to which a sialo-sugar-chain-containing compound was immobilized is disclosed, which method uses the property of HA protein to specifically adsorb to specific sugar chains. However, in this method, there are problems such as requirement of much labor in preparation of the carrier to which a sialo-sugar-chain-containing compound was immobilized, and probability of difficulty in the purification in cases where a large mutation occurred in the HA protein of the influenza virus strain.
In Non-patent Document 1, a method for preparation of HA protein, wherein a gene having information to allow production of HA protein of influenza virus is incorporated into animal cells (insect cells) by gene recombination to produce recombinant cells, which are then cultured, thereby producing HA protein, is described. This method requires thorough removal of impure proteins derived from the recombinant cells.